Altered tRNA dynamics during translocation on slippery mRNA as determinant of spontaneous ribosome frameshifting

When reading consecutive mRNA codons, ribosomes move by exactly one triplet at a time to synthesize a correct protein. Some mRNA tracks, called slippery sequences, are prone to ribosomal frameshifting, because the same tRNA can read both 0- and –1-frame codon. Using smFRET we show that during EF-G-catalyzed translocation on slippery sequences a fraction of ribosomes spontaneously switches from rapid, accurate translation to a slow, frameshifting-prone translocation mode where the movements of peptidyl- and deacylated tRNA become uncoupled. While deacylated tRNA translocates rapidly, pept-tRNA continues to fluctuate between chimeric and posttranslocation states, which slows down the re-locking of the small ribosomal subunit head domain. After rapid release of deacylated tRNA, pept-tRNA gains unconstrained access to the –1-frame triplet, resulting in slippage followed by recruitment of the –1-frame aa-tRNA into the A site. Our data show how altered choreography of tRNA and ribosome movements reduces the translation fidelity of ribosomes translocating in a slow mode.

In this study the authors use single-molecule FRET (smFRET) to follow the translocation of tRNALys through the ribosome in the context of a slippery mRNA sequence. By altering the placement of Cy3 and Cy5 fluorophores, they are able to follow the dynamics of EF-G catalyzed translocation relative to translocation on a non-slippery mRNA as a control. They show that translocation on a slippery sequence can proceed through two pathways. In one pathway, both A and P site tRNAs rapidly move to the P and E sites with no change in reading frame. In the other pathway the deacylated P-site tRNA rapidly translocates while the A-site peptidyl-tRNA is delayed at a late chimeric stage of translocation, where it fluctuates between chimeric (ap/P) and post-translocation (P/P) states. During this period the authors confirm that the small subunit head is swiveled, conditions where codon-anticodon pairing is destabilized and alternative pairing frames can be explored. In the case of spontaneous -1 frameshifting, rapid release of deacylated tRNA from the E-site following translocation may facilitate -1 pairing of the stalled peptidyl-tRNA. These insights into translocation on a slippery sequence help to explain how spontaneous -1 frameshifting occurs.
Central to this study is the demonstration of a positive correlation of -1 frameshifting on a slippery mRNA sequence to the fraction of peptidyl-tRNA that is delayed in translocation. This was accomplished by carrying out translocation in the presence of several mutants of EF-G containing amino acid substitutions at position 507. In the wild-type factor, glutamine at this position helps to stabilize codon-anticodon pairing of the peptidyl-tRNA. Substitutions of this amino acid are known to promote -1 frameshifting and the authors show that these same substitutions increase the fraction of translocation that is delayed due to fluctuation between ap/P and P/P states.
The smFRET studies appear to be carefully done. However, several minor revisions are needed. 1. As regards to the biochemical assay for -1 frameshifting, it is not specified whether peptide formation was carried out in the presence of both tRNAPhe and tRNAVal or only in the presence of the latter. By conducting this type of assay in the presence of both the 0-frame and -1-frame incoming tRNAs, any possibility of frameshifting in the P-site can be discounted. 2. The incorporation of 0-and -1 frame aa-tRNAs presented in Figure 3 does not add a lot to the paper and could be included in the supplemental section.

Answer to the REVIEWER COMMENTS
Reviewer #1 (Remarks to the Author): Manuscript by Poulis et al. presents additional smFRET look on the ribosome frameshifting associated with slippery sequences. Authors use EF-G mutants, different sets of slippery and non-slippery sequences, as well as slowly hydrolyzable GTPγS, fusidic acid and spectinomycin to test previously proposed model that slow frameshifting-prone translocation mode is responsible for reduced fidelity on slippery sequences. Reply: We politely disagree with the referee concerning the novelty and originality of our study. While previous work (reviewed in detail in the Introduction) has identified the putative link between the translocation rate and the tendency for the ribosome to frameshift, in this work we reveal the molecular mechanism on the single molecule level and additionally dissect the timing and the exact coupling between ribosome motions and frameshifting. Notably, in the previous work on programmed ribosome frameshifting slow translocation was induced by the downstream secondary structure element on the mRNA (which is not very surprising, as the ribosome encounters a hurdle that it struggles to pass). In contrast, here we study spontaneous frameshifting without such hurdles and show that an mRNA slippery sequence by itself changes the choreography of translocation, leading to frameshifting.
One of the control experiments " Figure 6 with wt EF-G on slippery sequence" requested by the referee was already present in our original text (Fig. 6a,b). We now performed the second control experiment concerning " Figure 3 with wt EF-G on slippery sequence"; the data are presented in the revised Fig. 3 and Supplementary Fig. 6 and 7. We show that also with EF-G(wt) ribosomes show slow translocation of pept-tRNA on slippery mRNA before accommodation of -1-frame Val-tRNA Val (Fig. 3e-h). However, since the majority of ribosomes stays in 0-frame with EF-G(wt) (Fig. 3a-d), -1-frame Val-tRNA Val is predominantly rejected . We never observed accommodation of -1-frame Val-tRNA Val after fast translocation of pept-tRNA, which makes prolonged fluctuations between CHI and P/P states an essential determinant of spontaneous frameshifting. We worked extensively on the data presentation in Fig. 3 and Supplementary Fig. 4, 5, 6, and 7. We simplified the cartoons, explained all abbreviations in the figure legends and replaced the term "TL" (translocation) for "CHI state", which was explained in the Introduction. The revised versions of Fig. 3 and Supplementary Fig.  4, 5,6, and 7 show that slow translocation via CHI and P/P states is a major determinant of spontaneous ribosome frameshifting.
We also added the data on the frameshifting efficiency by EF-G(Q507A) and EF-G(Q507N) mutants in Supplementary Fig. 3.
The proposed model in Figure 7b is based on EF-G mutant Q507D data and increased population of CHI and P/P states which are almost identical in GTPγS conditions (Fig 5), and on contrary to authors comments not really similar to spectinomycin data (in Supplementary Fig  8), which blurs authors conclusions. For these reasons I suggest that manuscript should not be published in this form.
Reply: We are surprised by this comment of the referee, because our transition frequency analysis clearly shows differences in translocation in the presence of EF-G(Q507D) and EF-G-GTPγS (compare Fig. 2e and Fig. 5d). EF-G(Q507D) fluctuates predominantly between FRET 0.4 and 0.2, whereas EF-G-GTPγS fluctuates between FRET 0.6 and 0.4, i.e. EF-G is stalled at a different step of translocation. The frameshifting efficiency is high during translocation with EF-G(Q507D) and pept-tRNA fluctuates between CHI and P/P states ( Fig. 2e and Suppelemtary Fig.  3). In contrast, the frameshifting efficiency during translocation by EF-G-GTPγS is low and tRNAs fluctuate predominantly between CHI and A/P* states (Fig. 5d). These results show that frameshifting occurs while ribosomes sample CHI and P/P states, whereas sampling of earlier intermediate states does not lead to frameshifting. Presumably, the referee is confused by the fact that contour plots with EF-G(Q507D) and EF-G-GTPγS look similar on first sight ( Fig. 2d and Fig. 5c). This is because these plots are dominated by the end levels (the P/P state) and were therefore further analyzed with respect to the transition frequency (Fig. 2e). In this analysis, we counted the number of transitions between FRET 0.6 (A/A, A/P*), FRET 0.4 (CHI) and 0.2 (P/P) states after EF-G binding to PRE complexes using idealized traces derived from the HMM fit of the FRET time course (see Methods). This information reveals in which step of the translocation pathway the movement of pept-tRNA is stalled (see also Adio et al. 2015). We added information in the Method section to better explain how the transition frequencies were derived.
The translocation pathway by EF-G-GTPγS is similar to that observed with EF-G(wt)-GTP in the presence of Spc (Fig. 5 and Supplementary Fig.11), as translocation is stalled at an early stage, consistent with other existing literature (Rundlet et al., Nature 2021, Belardinelli et al., RNA 2019and Adio et al., Nat Commun 2015. In both cases, pept-tRNA is stalled in fluctuations between A/P, A/P* and CHI states and transitions between CHI and P/P are practically absent. Translocation of deacylated tRNA is slow and occurs at the same rate as the translocation of pept-tRNA. We observed exactly the opposite behavior by EF-G(Q507D) (Fig. 2e, Fig.4e,f), where pept-tRNA is trapped in fluctuations between CHI and P/P, and translocation of deacylated tRNA is fast.
Our model (Fig. 7) shows the uncoupled movement of pept-and deacylated tRNA observed with the frameshifting-prone EF-G(Q507D) mutant. EF-G-GTPγS and Spc show only background levels of frameshifting (Fig. 5a), which is why these conditions serve as relevant controls but should not enter the main model.

Reviewer #2 (Remarks to the Author):
This is a very good manuscript which continues a longstanding effort by the Göttingen MPG group to fully elucidate the mechanism of -1 frameshifting. The major new results are the demonstration of two classes of ribosome during translation of slippery sequences, the correlation of the fraction of slow ribosome with frameshift efficiency, and the demonstration that tRNA dissociation from the E-site occurs more rapidly than the pep-tRNA fluctuation period. These results will be of interest to the community of researchers focused on the study of translation mechanisms. However, the points listed below need to be addressed before the MS would be acceptable for publication in Nature Communications.
MAJOR POINTS REQUIRING FURTHER CONSIDERATION: 1. Correlation of the fraction of slow ribosomes due to slippery sequences with frameshift efficiency. The results backing this conclusion are presented in Fig. 2f, for wt-EF-G and three variants. But the data referred to (Fig. S3) only include results for wt and one variant (Q507D). The results for the other two variants should also be shown in Fig. S3.
Reply: We added the data on the frameshifting efficiency by EF-G(Q507A) and EF-G(Q507N) mutants in Supplementary Fig. 3. 2. E-site occupancy during pep-tRNA fluctuation between CHI and P/P sites. The results presented on p. 7 clearly show that tRNA dissociation from the E-site occurs more rapidly than the fluctuation period, providing a "time window for pept-tRNA to switch to the -1-frame." This conclusion appears to be in conflict with the statement in the Discussion (p.11) that "frameshifting can occur both with one or two tRNAs bound, provided pept-tRNA is trapped in fluctuations between CHI and P/P." This apparent discrepancy should be further discussed.
Reply: We show that upon spontaneous -1-frameshifting, deacylated tRNA dissociates rapidly from the E site while pept-tRNA is still in the process of (slow) translocation (Fig. 1, 3 and 4). This indicates a one-tRNA slippage mechanism because the E-site codon is unoccupied while pept-tRNA has not reached the POST state. In contrast, during -1PRF (described in the discussion on p.12) the translocation of deacylated and pept-tRNA is coupled and occurs at similar rate (Caliskan et al., Cell 2014). Hence, depending on whether frameshifting occurs spontaneously or in a programmed manner, it can follow a one-or two-tRNA slippage mechanism. We modified the text (see top of p. 12) to point out that the two-tRNA slippage corresponds to -1PRF.
3. Insufficient documentation of results presented in Figs. 3b,h. The claimed transition from CR to AC is insufficiently documented in Fig. 3b. Contour plots, number of traces are needed. Similar comments for apply to transitions from CR in Fig. 3h. The lack of detail in Fig. 3h does not sufficiently support the two sentences beginning at the bottom of p.6 and continuing into p.7 " After some time, -1-frame Val-tRNAVal-Cy5 is accommodated into POST2 and then fluctuates between A/A, A/P and A/P* states. Accommodation of Val-tRNA provides a strong evidence that after the delayed translocation the ribosome moved into the -1-frame exposing the Val codon in the A site ( Fig. 3h-i)." Reply: We revised Fig. 3 and the corresponding figure legend. We explain the meaning of the abbreviation "CR" (codon recognition) and show that upon accommodation in the A site, tRNA Phe and tRNA Val fluctuate between classic (A/A) and hybrid (A/P and A/P*) conformations in a similar manner as pept-tRNA Lys (Supplementary Fig. 1). We included n=number of traces in the figure legend and compiled FRET signals reporting on the interaction of aa-tRNAs with POST2 complexes into contour plots (Fig. 3d,h,l and Supplementary Fig. 5d,h). The dwell time analysis of these FRET signals is now presented in the new Supplementary Fig. 6. The dissociation rates (koff) differ dramatically for the cognate aa-tRNA (which is accommodated) and the near-cognate tRNA (which is rapidly is rejected). Dissociation of -1-frame tRNA Val from PRE2 complexes formed after slow translocation on slippery mRNA is as slow as of the cognate 0-frame tRNA Phe on PRE2 complexes formed on non-slippery mRNA. This provides a strong evidence in favor of our conclusion that slowly-translocating ribosomes have moved into the -1-frame exposing the Val codon in the A site.
Please note that we also unified the experiment schemes in Fig. 3a, e, i. In all experiments, PRE1 complexes are immobilized and EF-G (wt or mutant) is added together with EF-Tu-aa-tRNA-GTP complex to induce tRNA accommodation and subsequent translocation. OTHER SIGNIFICANT POINTS: 1. A fuller description is needed of the significant structural differences between the CHI, hybrid and classical states than that currently provided in the Introduction. Given the importance of the CHI state, this will aid the understanding of the paper by all but the most knowledgeable readers.
Reply: We have added the description of the dynamics of PRE translocation complexes including explanation of tRNA conformations in hybrid and classical states. We have further explained that the CHI state is a transient intermediate state of translocation, which rapidly resolves into the POST state.
2. It is unclear when the EF-G is added in the TIRF microscopy experiments monitoring translocation (Figs 3b,e,h;4c,e;5b;6b). This point should be clearly addressed in either the Figure legends or the Experimental section.
Reply: In the experiments described in Fig. 3, EF-G was added to immobilized PRE1 complexes together with EF-Tu-GTP-aa-tRNA complex. The components were added with the imaging buffer approximately 10 s prior to imaging. We added additional description in the figure legends and also in the Methods section.
In the experiments described in Fig. 4, 5, 6 EF-G was added to immobilized PRE complexes approximately 10 s before imaging. This is now clearly stated in the Methods section.
Please note that the vertical line in Fig. 6 represents the synchronization point of the smFRET traces. This is now explained in the figure legend.
Reply: "The drift" in the Cy3 fluorescence signal of the example trace shown in our previous Fig.  3b represented noise in the data and did not correspond to tRNA movement. We replaced the trace for a different example trace with stable Cy3 fluorescence. 4. Fig. 4 -It is not clear from the data presented that the 0.9 -0.6 transition represents a fluctuation, as opposed to an essentially unidirectional transition from 0.9 to 0.6. Contour plots should be provided for interconversions beween the 0.9, 0.6 and 0.3 FRET states Reply: We changed the text (p. 7) to clarify that the transitions between 0.9 and 0.6 states are not unidirectional but much less frequent than fluctuations of pept-tRNA on PRE complexes. Additionally, we calculated the transition frequency of deacylated tRNA (i.e. the average number of transitions between 0.6 and 0.9 FRET per trace) and compared it with the transition frequency of pept-tRNA on PRE complexes. Values differ by about one order of magnitude (0.4 vs 5.4 transitions, Supplementary Fig. 1c and 8f), further supporting the point that fluctuations of deacylated tRNA on PRE complexes are relatively slow. Transitions into the POST (0.3 FRET) state are irreversible ( Supplementary Fig. 9d) and occur from the PRE state with either FRET 0.9 or 0.6, which is shown in the contour plot (Fig. 4d,e). Supplementary Figures